Download ELUCIDATION OF A PERIBACTEROID MEMBRANE

ELUCIDATION OF A PERIBACTEROID
MEMBRANE-BOUND bHLH
TRANSCRIPTION FACTOR REQUIRED
FOR LEGUME NITROGEN FIXATION
A THESIS SUBMITTED BY
PATRICK CHARLES LOUGHLIN
SCHOOL OF AGRICULTURE, FOOD AND WINE
UNIVERSITY OF ADELAIDE
NOVEMBER 2007
I Abstract
Many legumes, including soybean, are agriculturally important crop plants. Legumes
are unique in their ability to form an endo-symbiosis with soil borne bacteria
collectively called rhizobia, which allows the plant to access atmospheric di-nitrogen
via the bacteria. The interface between the legume and differentiated, intracellular
rhizobia (called bacteroids) is a plant derived membrane called the peribacteroid
membrane (PBM). This membrane has a unique complement of proteins, which are
required to maintain the bacteroids’ environment and allow bi-directional transport of
solutes. One such PBM protein from soybean is GmSAT1 (Glycine max symbiotic
ammonium transporter 1) which was initially characterised as a PBM-localised
ammonium transporter based on its ability to complement an ammonium transportdeficient yeast strain 26972c (Kaiser et al., 1998). Subsequent research however, has
suggested that GmSAT1 is not directly involved in ammonium transport (Marini et
al., 2000).
This project sought to shed some light on the functional role of this intriguing protein.
GmSAT1 is unusual in that it has both high homology with known transcription
factors of the basic Helix-Loop-Helix (bHLH) family, as well as a predicted Cterminal transmembrane domain. Conservative amino acid substitutions within the
bHLH transcription factor domain of GmSAT1 completely abolished the ability of the
protein to complement growth of the ammonium transport-deficient yeast 26972c on
low ammonium medium. The localisation of GmSAT1 in both soybean and a yeast
expression system were examined in depth using immunolocalisation, western
blotting of subcellular protein fractions, and GFP fusion proteins. Immunogold
ii
labelling of rhizobia-infected nodule cells verified the localisation of GmSAT1 to the
PBM (Kaiser et al., 1998) and additionally the protein was localised in the nucleus.
Western blotting demonstrated that GmSAT1 is present as two different size proteins
in soybean nodules, with the full length protein present in the insoluble fraction and a
truncated protein present in the soluble protein fraction. Biochemical evidence in
yeast using a modified two-hybrid reporter system suggests that the GmSAT1 protein,
either the full length protein or the N-terminal part, is localised to the nucleus.
GmSAT1-GFP fusion protein was localised to small punctate bodies around the yeast
cell, adjacent to the plasma membrane and in some instances co-localised with a
nuclear stain, also suggesting nuclear localisation.
A soybean genetic transformation protocol was developed to examine the role of
GmSAT1 in the symbiosis between soybean and Bradyrhizobium japonicum through
RNAi gene silencing. Results suggest that GmSAT1 is essential for normal nodule
development, with GmSAT1-silenced (sat1) nodules being smaller and ineffective in
providing the soybean plant with sufficient fixed nitrogen. Rhizobia-infected cells in
sat1 nodules were distinctive in that they retained central vacuoles and did not
increase in size and consequently there were far fewer bacteroid located in these cells.
Taken together, our results suggest that GmSAT1 is a membrane-bound transcription
factor, located in rhizobia-infected nodule cells of soybean. Upon an as yet
undetermined signal, GmSAT1 is proteolytically cleaved from the membrane and
imported into the nucleus to activate gene transcription. The functional role of
GmSAT1 in planta is yet to be determined, however silencing data suggest that it is
essential for the maintenance of effective nodules.
iii
II Declaration
This work contains no material which has been accepted for the award of any other
degree or diploma in any university or other tertiary institution and, to the best of my
knowledge and belief, contains no material previously published or written by another
person, except where due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library,
being made available for loan and photocopying, subject to the provisions of the
Copyright Act 1968.
Patrick Charles Loughlin
November 2007
iv
III Acknowledgements
Numerous people have assisted me over the course of my PhD. There are those who
have assisted directly in my scientific endeavours, and indirectly in keeping me on a
relatively even keel, and those that have helped in both. My thanks go to my
supervisors Brent Kaiser and Steve Tyerman, who took me on nearly four years ago
now and hopefully they haven’t regretted it. Brent in particular has helped me when
results were looking grim having the knack of turning a seemingly negative result into
something positive. He has both guided me when I needed it and left me to my own
devices when I didn’t, and for this I am grateful. Thanks also go to the Kaiser and
Tyerman lab members, both past and present, where at times it felt a bit like the blind
leading the blind, but we got there in the end. Without the scientific support and
camaraderie of people like Scott Carter, Kate Gridley, Megan Shelden, Rebecca
Vandeleur, Sunita Ramesh, Cass Collins, Christian Preuss, Wendy Sullivan, Jess
Parker and Toni Cordente I don’t think I would have made it. Special thanks to Scott
Carter who was always willing to lend a hand in the lab when it was needed, or have a
coffee (or something stronger) with me when that was needed. The initial technical
assistance in confocal and transmission electron microscopy by Peter Kolesik and the
Adelaide Microscopy team was much appreciated. During my PhD I was also given
the opportunity to spend two months in the lab of Prof. Tony Glass at the University
of British Columbia in Vancouver. This experience was enlightening, both
scientifically and personally, so I wish to thank Tony, WenBin, and Ye for making me
feel welcome there. Finally I would like to acknowledge the moral support and love
from my family and friends over the course of my PhD, without which, the whole
experience would have been a whole lot tougher.
v
IV Abbreviations
bHLH
Basic Helix-Loop-Helix
BLAST
Basic local alignment tool
BSA
Bovine serum albumin
cDNA
Complementary DNA
CaMV
Cauliflower mosaic virus
Cub
C-terminal ubiquitin
ddH2O
Double distilled H2O
dsRNA
Double stranded RNA
DTT
Dithiothreitol
EDTA
Ethylenediaminetetraacetic acid (disodium salt)
EMS
Ethylmethane sulfonate
ER
Endoplasmic reticulum
GES
Goldman-Engelmen-Steitz
GFP
Green fluorescent protein
GUS
E-glucoronidase
HIS
Histidine
hpRNA
Hairpin RNA
kB
Kilobase
kDa
Kilodalton
LB
Luria broth (medium)
LZ
Leucine zipper
MA
Methylammonium (chloride)
MeJA
Methyl jasmonic acid
MES
2-[N-Morpholino]ethanesulfonic acid
MW
Molecular weight
NCR
Nitrogen catabolite repression
Nub
N-terminal ubiquitin
OD
Optical density
ORF
Open reading frame
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PBM
Peribacteroid membrane
PBS
Peribacteroid space OR Phosphate buffered saline
PCR
Polymerase chain reaction
PEG
Polyethylene glycol
PMSF
Phenylmethylsulfonyl fluoride
Pro
Proline
RPM
Revolutions per minute
RNA
Ribonucleic acid
RNAi
RNA interference
rRNA
ribosomal RNA
SDS
Sodium dodecyl sulfate
SDS-PAGE
SDS polyacrylamide gel electrophoresis
ssDNA
Salmon sperm DNA
TAE
Tris acetate EDTA
TBS
Tris buffered saline
TCA
Trichloroacetic acid
TE
Tris EDTA
TF
Transcription factor
TMD
Transmembrane domain
Tris
Tris(hydroxymethyl)aminomethane
UPR
Unfolded protein response
UV
Ultraviolet
v/v
Volume/volume
w/v
Weight/volume
X-gal
5-bromo-4-chloro-3-indolyl-ȕ-D-galactopyranoside
X-gluc
5-bromo-4-chloro-3-indolyl-ȕ-D-glucoronic acid
YEM
Yeast extract mannitol (medium)
YNB
Yeast nitrogen base (medium)
YPAD
Yeast extract peptone adenine dextrose (medium)
vii
Table of Contents
I.
ABSTRACT……………….……………………………………………..ii
II.
DECLARATION……………………………………………………...…iv
III.
ACKNOWLEDGEMENTS………………………………………………v
IV.
ABBREVIATIONS……………………………………………………...vi
1. Introduction..................................................................................... 1-1
1.1 INTRODUCTION.............................................................................................. 1-1
1.2 NODULATION .................................................................................................. 1-2
1.2.1 SIGNAL EXCHANGE AND NODULE DEVELOPMENT.................................................. 1-2
1.3 THE PERIBACTEROID MEMBRANE ......................................................... 1-5
1.3.1 BIOGENESIS OF THE PERIBACTEROID MEMBRANE .................................................. 1-5
1.3.2 PROTEIN TRAFFICKING TO THE PBM............................................................................ 1-6
1.4 FUNCTIONALLY CHARACTERISED PBM ENZYMES AND CARRIERS
.................................................................................................................................... 1-7
1.4.1 H+-ATPASE AND OTHER PBM-ASSOCIATED ENZYMES............................................. 1-7
1.4.2 NOD26.................................................................................................................................... 1-8
1.4.3 PBM ION TRANSPORT........................................................................................................ 1-9
1.4.4 EXCHANGE OF FIXED CARBON AND NITROGEN ACROSS THE PBM................... 1-10
1.4.4.1 CARBON FLUX ACROSS THE PBM ........................................................................... 1-10
1.4.4.2 NITROGEN FLUX ACROSS THE PBM ....................................................................... 1-11
1.4.4.3 CHARACTERISATION OF THE PBM AMMONIUM CARRIER ................................. 1-12
1.5 GmSAT1 ........................................................................................................... 1-13
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1.5.1 FUNCTIONAL CHARACTERISATION OF GmSAT1 ..................................................... 1-13
1.5.2 GmSAT1 AND YEAST AMMONIUM TRANSPORT....................................................... 1-14
1.5.3 IS GmSAT1 A TRANSCRIPTION FACTOR?.................................................................... 1-14
1.6 MEMBRANE-BOUND TRANSCRIPTION FACTORS............................. 1-15
1.6.2 PLANT MEMBRANE-BOUND TRANSCRIPTION FACTORS....................................... 1-17
1.7 CHARACTERISED NODULE TRANSCRIPTION FACTORS................ 1-18
1.8 PROJECT AIMS ............................................................................................. 1-19
FIGURES............................................................................................ 1-21
Figure 1-1. The soybean symbiosome ............................................ 1-21
2. A Functional Analysis of GmSAT1 using Site-Directed
Mutagenesis............................................................................................ 2-1
2.1 INTRODUCTION.............................................................................................. 2-1
2.1.1 THE BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTOR FAMILY..................... 2-1
2.1.2 THE FUNCTION OF GmSAT1 AND STRUCTURAL ASPECTS OF ITS bHLH MOTIF. 2-2
2.2 RESULTS ........................................................................................................... 2-4
2.2.1 MUTATIONS IN THE TRANSMEMBRANE DOMAIN OF GmSAT1 HAVE MIXED .... 2-4
EFFECTS ON ITS FUNCTION...................................................................................................... 2-4
2.2.2 THE BASIC REGION OF THE bHLH DOMAIN OF GmSAT1 IS ESSENTIAL FOR
FUNCTION ..................................................................................................................................... 2-5
2.2.3 MUTATION OF HYDROPHOBIC RESIDUES IN THE HELICES OF THE bHLH
DOMAIN ALSO AFFECT GmSAT1 FUNCTION ........................................................................ 2-7
2.2.4 OVEREXPRESSION OF ENDOGENOUS YEAST bHLH TRANSCRIPTION FACTORS
AND NITROGEN METABOLISM-RELATED PROTEINS DO NOT MIMIC GmSAT1
EXPRESSION ................................................................................................................................. 2-8
ix
2.3 DISCUSSION ................................................................................................... 2-11
2.4 METHODS ....................................................................................................... 2-15
2.4.1 PCR AMPLIFICATION OF MUTANT GmSAT1 cDNA .................................................... 2-15
2.4.2 SELECTION OF MUTANT GmSAT1 ................................................................................. 2-16
2.4.3 YEAST TRANSFORMATION............................................................................................ 2-16
2.4.4 GROWTH STUDIES OF 26972C EXPRESSING MUTANT GmSAT1 ............................ 2-17
2.4.5 14C-METHYLAMMONIUM FLUX .................................................................................... 2-18
2.4.6 TOTAL YEAST PROTEIN EXTRACTION FOR SDS/PAGE........................................... 2-18
2.4.7 SODIUM DODECYL SULFATE/ POLYACRYLAMIDE GEL ELECTROPHORESIS... 2-19
2.4.8 WESTERN BLOTTING....................................................................................................... 2-19
TABLES.............................................................................................. 2-21
Table 2-1. Primers used to introduce point mutations into the bHLH
and TMD of GmSAT1..................................................................... 2-21
FIGURES............................................................................................ 2-22
Figure 2-1. A model of the putative homodimeric bHLH domain of
GmSAT1 bound to DNA ................................................................. 2-22
Figure 2-2. Sequence alignments of the bHLH domain of GmSAT1
(Q169 o M217) and selected bHLH proteins ............................... 2-23
Figure 2-3. GmSAT1 has a predicted C-terminal transmembrane
domain (TMD)................................................................................. 2-24
Figure 2-4. Effects of amino acid substitutions in the predicted Cterminal transmembrane domain of GmSAT1 .............................. 2-25
Figure 2-5. The effects of mutations in the basic region of the
GmSAT1 bHLH domain ................................................................. 2-26
Figure 2-6. The effects of mutations in helix 1 of the GmSAT1
bHLH domain ................................................................................. 2-27
Figure 2-7. The effects of mutations in helix 2 of the GmSAT1
bHLH domain ................................................................................. 2-28
Figure 2-8. Sequence alignment (A) and phylogenetic tree (B) of the
basic Helix-Loop-Helix domain of plant members of the SAT family
.......................................................................................................... 2-29
x
Figure 2-9. Sequence alignment of GmSAT1 with the yeast bHLH
and bHLH-like proteins (boxed) used in this study ....................... 2-30
Figure 2-10. Map of yeast ORF overexpression shuttle vector
BG1805 (Open Biosystems) ............................................................ 2-31
Figure 2-11. Growth studies of 26972c yeast overexpressing
endogenous bHLH and nitrogen catabolite repression- (NCR)
associated genes .............................................................................. 2-32
3. Subcellular Localisation of GmSAT1 ............................................ 3-1
3.1 INTRODUCTION.............................................................................................. 3-1
3.1.1 MEMBRANE-BOUND TRANSCRIPTION FACTORS....................................................... 3-2
3.2 RESULTS ........................................................................................................... 3-3
3.2.1 GmSAT1 IS PRESENT AS TWO DIFFERENT SIZE PROTEINS IN THE SOYBEAN
NODULE AND YEAST ................................................................................................................. 3-3
3.2.2 GmSAT1 LOCALISES TO BOTH INTERNAL MEMBRANE SYSTEMS AND NUCLEI OF
INFECTED NODULE CELLS ....................................................................................................... 3-5
3.2.3 GmSAT1 IS LOCALISED TO PUNCTATE, CELL PERIPHERAL VESICLES AND THE
NUCLEUS OF YEAST................................................................................................................... 3-6
3.2.4 A YEAST TWO-HYBRID ASSAY PROVIDES BIOCHEMICAL EVIDENCE THAT THE
N-TERMINAL PART OF THE GmSAT1 PROTEIN IS LOCALISED TO THE NUCLEUS ...... 3-7
3.2.5 MUTATION OF A PUTATIVE PROTEOLYTIC CLEAVAGE SITE IN GmSAT1
ABROGATES SELF ACTIVATION OF REPORTER GENE EXPRESSION.............................. 3-8
3.2.6 MUTATIONS IN THE PUTATIVE PROTEOLYSIS MOTIF AFFECT GmSAT1
FUNCTION IN YEAST .................................................................................................................. 3-9
3.2.7 MODULATION OF THE EXPRESSION LEVELS OF LEU277 GmSAT1 MUTANTS
REVEALS FURTHER FUNCTIONAL DIFFERENCES WHEN COMPARED WITH WILDTYPE GmSAT1............................................................................................................................. 3-10
3.3 DISCUSSION ................................................................................................... 3-11
xi
3.3.1 GmSAT1 IS PROBABLY CLEAVED AT THE SAME SITE IN BOTH YEAST AND
SOYBEAN .................................................................................................................................... 3-11
3.3.2 THE DUAL LOCALISATION OF GmSAT1 WHEN EXPRESSED IN YEAST............... 3-13
3.3.3 CLEAVAGE OF GmSAT1 FROM SOYBEAN AND YEAST MEMBRANES? ............... 3-14
3.4 METHODS ....................................................................................................... 3-16
3.4.1 VECTOR CONSTRUCTION............................................................................................... 3-16
3.4.2 PCR AMPLIFICATION OF ‘RXXL’ MUTANT GmSAT1 ................................................. 3-18
3.4.3 YEAST TECHNIQUES ....................................................................................................... 3-18
3.4.4 LACZ ACTIVITY DETERMINATION IN YEAST ........................................................... 3-18
3.4.5 DELETION OF LEU2 FROM THE 26972C GENOME ..................................................... 3-19
3.4.6 CONFOCAL FLUORESCENCE MICROSCOPY .............................................................. 3-20
3.4.7 EXTRACTION AND SEPARATION OF SOLUBLE AND INSOLUBLE PROTEIN YEAST
FRACTIONS ................................................................................................................................. 3-20
3.4.8 EXTRACTION AND SEPARATION OF SOLUBLE AND INSOLUBLE PROTEIN
FRACTIONS FROM SOYBEAN NODULES.............................................................................. 3-21
3.4.9 IMMUNOGOLD LABELLING OF SOYBEAN NODULE SECTIONS............................ 3-22
TABLES.............................................................................................. 3-23
Table 3-1. Primers used in Chapter 3............................................. 3-23
Table 3-2. Primers used to introduce point mutations into the
‘RXXL’ putative proteolytic cleavage site of GmSAT1.................. 3-24
FIGURES............................................................................................ 3-25
Figure 3-1. Anti-GmSAT1 serum recognises at least two different
size epitopes in both soybean nodules and yeast expressing GmSAT1
.......................................................................................................... 3-25
Figure 3-2. Immunogold-localisation of GmSAT1 to the
peribacteroid membrane of infected nodule cells .......................... 3-26
Figure 3-3. Immunogold-localisation of GmSAT1 to the nucleus and
PBM of infected nodule cells.......................................................... 3-27
Figure 3-4. GmSAT1 antiserum is necessary to localise immunogoldconjugated IgG to the nucleus of infected nodule cells................. 3-28
xii
Figure 3-5. Construction of yeast GFP expression vectors........... 3-29
Figure 3-6. GFP-fusions of GmSAT1 are functional ................... 3-30
Figure 3-7. Localisation of GFP expressed alone or fused to the Cterminus or N-terminus of GmSAT1 expressed in yeast ............... 3-31
Figure 3-8. N-terminal GFP-GmSAT1 fusion protein localises to
small punctate bodies and the nucleus of yeast ............................. 3-32
Figure 3-9. The Dualsystems membrane protein yeast two hybrid
system (taken from www.dualsystems.com) ................................... 3-33
Figure 3-10. Activation of reporter gene expression in DSY-1 yeast
expressing GmSAT1 fused with an N-terminal but not C-terminal
artificial transcription factor .......................................................... 3-34
Figure 3-11. Comparison of the protein structures of GmSAT1 with
mammalian membrane-bound transcription factors cleaved by the
Site-1 protease ................................................................................. 3-35
Figure 3-12. The effects of various amino acid substitutions in
GmSAT1 on HIS3 reporter gene expression ................................. 3-36
Figure 3-13. Effects of amino acid substitutions in the putative
proteolysis site of GmSAT1............................................................. 3-37
Figure 3-14. Modulating GmSAT1 expression levels reveals
differences between wild-type and L277 mutants in their ability to
complement 26972c on 1 mM NH4................................................. 3-38
4.
Genetic Transformation of Soybean and Silencing of GmSAT1
and GmNOD26....................................................................................... 4-1
4.1 INTRODUCTION.............................................................................................. 4-1
4.1.1 GENETIC TRANSFORMATION OF PLANTS ................................................................... 4-1
4.1.2 SELECTION OF TRANSFORMED PLANT TISSUE.......................................................... 4-2
4.1.3 GENE SILENCING IN PLANTS .......................................................................................... 4-3
4.1.4 GmSAT1 AND OTHER NODULE-EXPRESSED GENES .................................................. 4-4
4.2 RESULTS ........................................................................................................... 4-5
xiii
4.2.1 TRANSFORMED SOYBEAN ROOTS ARE EFFICIENTLY PRODUCED THROUGH
INOCULATION OF RADICLE WOUND SITE WITH A. rhizogenes K599 ................................ 4-5
4.2.2 GUS EXPRESSION UNDER THE CaMV35S PROMOTER IS SYSTEMIC THROUGHOUT
SOYBEAN ROOTS AND NODULES ........................................................................................... 4-6
4.2.3 GUS EXPRESSION DRIVEN BY THE Gmlbc3 PROMOTER IS PREDOMINANTLY
RESTRICTED TO THE INFECTED INNER CORTEX OF THE NODULE................................ 4-7
4.2.4 VERIFICATION OF THE TRANSFORMATION OF SOYBEAN ROOTS WITH
pHELLSGATE 8 SILENCING VECTORS .................................................................................... 4-8
4.2.5 GmSAT1 IS ESSENTIAL FOR NODULE DEVELOPMENT AND/OR MAINTENANCE 4-9
4.2.6 SILENCING OF GmNOD26 IN SOYBEAN ROOTS......................................................... 4-10
4.3 DISCUSSION ................................................................................................... 4-11
4.3.1 DEVELOPMENT OF AN EFFICIENT SOYBEAN TRANSFORMATION PROTOCOL 4-11
4.3.2 BOTH THE CaMV35S AND Gmlbc3 PROMOTERS ARE ACTIVE IN A. rhizogenes
TRANSFORMED SOYBEAN TISSUE ....................................................................................... 4-13
4.3.3 SILENCING OF GmSAT1 AND GmNOD26 HAVE DIFFERENT EFFECTS ON THE
SOYBEAN-BRADYRHIZOBUM SYMBIOSIS ............................................................................ 4-14
4.4 METHODS ....................................................................................................... 4-16
4.4.1 VECTOR CONSTRUCTION............................................................................................... 4-16
4.4.2 PREPARATION AND TRANSFORMATION OF CHEMICALLY COMPETENT A.
rhizogenes K599 ............................................................................................................................ 4-17
4.4.3 ‘HAIRY ROOT’ A. rhizogenes-MEDIATED TRANSFORMATION OF SOYBEAN ....... 4-18
4.4.4 GENOMIC DNA ISOLATION AND TRANSFORMATION VERIFICATION................ 4-19
4.4.5 GUS STAINING OF SOYBEAN ROOT AND NODULE TISSUE.................................... 4-20
4.4.6 MICROSCOPY .................................................................................................................... 4-20
TABLES.............................................................................................. 4-22
Table 4-1. PCR primers used to amplify gene fragments for
pHellsgate8 silencing vector ........................................................... 4-22
FIGURES............................................................................................ 4-23
xiv
Figure 4-1. pCAMBIA (www.cambia.org) binary vectors (A-D) used
in this study...................................................................................... 4-23
Figure 4-2. RNAi silencing vectors used in this study................... 4-24
Figure 4-3. Soybean A. rhizogenes- mediated hairy root
transformation (adapted from Boisson-Dernier etal., 2001)......... 4-25
Figure 4-4. Inhibitory effects of hygromycin on soybean growth 4-26
Figure 4-5. CaMV35S promoter-driven GUS expression in soybean
roots and nodules ............................................................................ 4-27
Figure 4-6. GUS expression under the soybean leghaemoglobin
promoter (Gmlbc3).......................................................................... 4-28
Figure 4-7. Confirmation of transformation of roots with
pHellsgate8-derived vectors ............................................................ 4-29
Figure 4-8. Phenotype of GmSAT1-silenced (sat1) roots and nodules
.......................................................................................................... 4-30
Figure 4-9. Microscopic analysis of structural aberrations of sat1
nodules............................................................................................. 4-31
Figure 4-10. The phenotype of putatively GmNOD26-silenced
soybean plants ................................................................................. 4-32
5. General Discussion ......................................................................... 5-1
5.1 THE LEGUME-RHIZOBIA SYMBIOSIS ..................................................... 5-1
5.2 INITIAL IDENTIFICATION AND CHARACTERISATION OF GmSAT1 52
5.3 GmSAT1 ACTS AS A bHLH TRANSCRIPTION FACTOR ....................... 5-4
5.4 THE MEMBRANE LOCALISATION OF GmSAT1 .................................... 5-5
5.5 GmSAT1: A MEMBRANE BOUND TRANSCRIPTION FACTOR ........... 5-6
5.5.1 THE ENDO-PROTEOLYSIS OF GmSAT1 .......................................................................... 5-7
5.5.2 CLEAVAGE OF GmSAT1 BY A B. japonicum ENCODED PROTEASE? ....................... 5-10
xv
5.6 GmSAT1 AND ITS ROLE IN AMMONIUM / METHYLAMMONIUM
TRANSPORT IN 26972c....................................................................................... 5-10
5.6.1 GmSAT1 INCREASES Mep3 EXPRESSION IN 26972C .................................................. 5-10
5.6.2 THE ROLE OF GmSAT1 IN MA ACCUMULATION AND TOXICITY IN YEAST....... 5-11
5.7 GmSAT1 IS ESSENTIAL FOR SYMBIOSME DEVELOPMENT ........... 5-15
5.7.1 A ROLE FOR GmSAT1 IN TRAFFICKING OF PROTEINS TO THE PBM? .................. 5-16
5.7.2 GmSAT1 AND SOYBEAN NODULE NITROGEN BALANCE ....................................... 5-17
5.8 IDENTIFICATION OF A GMSAT1-LIKE GENE FROM SOYBEAN..... 5-17
5.9 SUMMARY ...................................................................................................... 5-18
FIGURES............................................................................................ 5-20
Figure 5-1. Three possible mechanisms by which GmSAT1
expression causes MA toxicity in 26972......................................... 5-20
Figure 5-2. A model depicting the role of GmSAT1 in the soybean
infected nodule cell ......................................................................... 5-21
Figure 5-3. Protein sequence alignment of GmSAT1 and GmSAT2 522
6. References ....................................................................................... 6-1
APPENDIX A...........................................................................................a
Table A1. Yeast strains used in the appendices ...................................a
Figure A1. GmSAT1 expression increases Mep3 transcript
abundance in 26972c ............................................................................b
Figure A2. Mep3 does not contribute to MA uptake ...........................c
Figure A3. Mep3 transports sufficient ammonium to allow growth on
1mM ammonium media but does not cause toxicity when grown in
100 mM MA...........................................................................................d
xvi